Chromatography, for example liquid chromatography (LC), gas chromatography (GC) or supercritical fluid chromatography (SFC), is employed in both analytical and preparative methods to separate one or more species, e.g., chemical compounds, present in a carrier phase from the remaining species in the carrier phase. Chromatography is also employed, in a manner independent of separation of chemical species, as a method for analyzing purity of a chemical specie, and/or as a means of characterizing a single chemical specie. Characterization of a chemical specie may comprise data, for example, a retention time for a particular chemical compound, when it is eluted through a particular chromatography column using specified conditions, e.g., carrier phase composition, flow rate, temperature, etc. These instruments may generally comprise a reservoir of mobile phase (which may be a gas, a liquid or supercritical fluid), a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting an aliquot of the sample mixture into the column. The different components of the mixture pass through the separation column at different rates due to differences in their respective mobilities between the mobile phase and the stationary phase. For example, a component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these mobility differences, sample components are separated from each other as they travel through the stationary phase. The instrument may then elute the solutes to a flow-through detector, such as a mass spectrometer. The solutes may, alternatively, pass to a collection system for later use, further analysis, or successive separation.
For reversed phase liquid chromatography systems, the mobile phase typically comprises water and one or more water-miscible organic modifiers, for example, acetonitrile or methanol. The specie-of-interest typically forms a solution with the mobile phase. Affinity of a specie for a stationary phase, which affects the rate at which a particular specie in a carrier phase passes through a stationary phase, results primarily from interaction of the specie with chemical groups present on the stationary phase. Chemical groups may be provided on the stationary phase by reacting a surface-modifying reagent with a substrate, such as a silica substrate. Surface-modifying agents may thus be employed to adsorb specific chemical groups onto the stationary phase. There is a continuing need in the art for improved chromatographic methods for the separation of eluent species.
Processing of biological materials may involve the use of liquid chromatography to separate and harvest cellular product or metabolite of interest from cellular debris or cell media. The separated biological components may be collected post-separation and thus fractionated for purposes ranging from protein purification or sample reduction prior to other downstream analyses. Generally, the eluent may be monitored using an ultraviolet-visible (UV-VIS) light-range spectrophotometer, and fractions which are thought to contain the product of interest are collected and sometimes pooled. The fractions may be further analyzed using any of a variety of biochemical analyses, such as Western blotting, SDS-PAGE, ELISAs, protein sequencing, or the like, in order to determine the presence of the target analyte(s), quantify the identified analyte(s), or both.
Some of the most important cellular functions, including cell signaling, are implicated in a number of proteins tied to biological membranes, e.g., lipid rafts. Lipid rafts, also referred to as detergent resistant membrane fragments, are localized cell membrane regions, or subdomains, enriched in cholesterol, glycosphingolipids, and integral membrane proteins. See e.g., Foster et al., Proc. Natl. Acad. Sci. USA 100(10):5813-8 (2003); also Simons et al., Nat. Rev. Mol. Cell Biol. 1(1):31-9 (2000). These heterogeneous membrane fragments were originally associated with lipid trafficking, but now appear to be implicated in a variety of biological processes, such as signal transduction, endocytosis, protein processing and pathogen entry. See e.g., Becher et al., Biochem. Biophys. Res. Commun. 321(4):981-7 (2004); also Manie et al., J. Virol. 74(1):305-11 (2000). However, proteomic samples, such as human serum, or lipid rafts are particularly complex and challenging to isolate and separate due to the large range of protein concentrations. Moreover, lipids present in biological extracts may bind to proteins and interfere with protein detection, quantization, or both. As a result, membrane proteins are generally less studied than soluble proteins due to the numerous impediments encountered in the delipidation, protein separation and structure/function analyses of this protein class.
Lipid rafts, nonetheless, are a readily accessible source of membranous material enriched in integral membrane proteins, and as such, provide a good starting material to define separations conditions that have broad utility. Lipid rafts are commonly prepared from cultured cells and disrupted tissues by selective solubilization with detergents, followed by differential centrifugation. Significant dependencies on protein and lipid compositions of such preparations are seen, based on the conditions used for detergent extraction and sample workup. See e.g., Pike, J. Biochem. J. 378 (Pt 2):281-92 (2004).
Traditionally, proteomic analyses of complex protein samples involve the resolution of proteins using two-dimensional gel electrophoresis (2DGE), followed by the identification of resolved proteins by mass spectrometry or simply by shotgun proteomics methods, which combine two dimensional LC and mass spectrometry. However, by either method, solubility and recovery of proteins remains an obstacle. For 2D gel electrophoresis, many hydrophobic proteins are not solubilized in the non-detergent isoelectric focusing sample buffer and solubilized proteins are prone to precipitation at their isoelectric point. For shotgun proteomic methods, as well as 2DGE, limited dynamic range of detection is also at issue because membrane proteins are typically lower in abundance when compared with soluble proteins. To resolve this problem, lipids are often solubilized using detergents in order to delipidate the associated proteins. However, certain chromatographic methods, such as reversed phase-HPLC (RP-HPLC), poorly tolerate the presence of detergent in samples, and become all but inoperable with the addition of detergent to the mobile phase. In fact, RP-HPLC separations of complex protein samples generally have low sample recoveries, and provide poor reproducibility and inadequate resolution. Consequently, chromatography of high-molecular-mass hydrophobic proteins presents a myriad of challenges that often prohibit their utility and impede investigation.
Alternatively, samples may be delipidated using an organic modifier or a mixture of organic modifiers. For example, a common delipidating procedure for plasma, protein solutions, cell culture, or disrupted tissue involves the use of a mixture of butanol and di-isopropyl ether or acetone or ethanol precipitation. Using these methods, the proteins precipitate while the organic phase retains the dissolved lipids. Many of these methods also generally require further centrifugation and extraction steps. Still further, these typical dilapidation methods generally exhibit several failings including protein losses, and solubility or miscibility concerns, making it difficult to fully recover proteins or lipids of interest. Indeed, protein losses and method reproducibility, remain a significant problem in the art.
Considerable research has, therefore, been directed toward the development of chromatographic delipidation methods suitable for protein-containing mixtures, such as biological fluids. For example, U.S. Pat. No. 5,885,921 discloses hydrophobic silica that may be suitable for the adsorption of lipids from whole blood. As stated in the '591 patent, however, such a product is not suitable as a chromatographic support media. Thus, there remains a need in the art for delipidation methods, including on-column methods, suitable for use with lipid-containing mixtures having high lipid content and/or high protein content. Further, there exists a need for on-column delipidation techniques that minimizes protein losses and/or improves sample processing and reproducibility.